The long-range goal of this research is to identify bioactive components and develop novel nutraceuticals that may improve vascular function and therefore decrease the morbidity and mortality related to vascular dysfunction and related complications. Vascular inflammation and its subsequent endothelial dysfunction play a fundamental role in the initiation and progression of atherosclerosis. It is believed that tumor necrosis factor (TNF)-alpha is critically involved in the pathogenesis of atherosclerosis. Studies show that genistein may have vascular protective effects. However, the molecular mechanism of genistein action is not well understood. The main objective of this study is to evaluate the cellular mechanism of action of genistein in its protective effect on (TNF)-alpha-induced vascular dysfunction. We have collected preliminary data, which suggest a potential anti-inflammatory action of genistein against vascular dysfunction: 1) genistein, at physiological concentrations, significantly inhibited TNF-alpha-induced adhesion of monocytes to human umbilical vein endothelial cells (HUVECs), 2) genistein significantly suppressed TNF-alpha-induced production of monocyte chemoattractant protein -1 (MCP-1) and interleukin-8 (IL-8), two chemokines that are the key factors in the firm adhesion of monocytes to activated endothelial cells, (3) TNF-alpha significantly increased NF-KB binding activity indicating that activation of the transcription factor NF-kB might be critical for the TNF-alpha-induced inflammatory response. We have recently demonstrated that genistein directly activates the cAMP/protein kinase A (PKA) signaling in endothelial cells, which is not related to any known genistein action such as inhibition of tyrosine kinase or binding to estrogen receptors. Our working hypothesis is that genistein, at physiological concentrations, suppresses TNF-alpha-induced recruit of monocytes to endothelial cells via activation of the cAMP/PKA signaling (involving modulation of plasma membrane associated G-proteins) that subsequently inhibits NF-kB activity and its regulated chemokine and adhesion molecular expression. Accordingly, the specific aims of this application are: 1) to determine whether genistein inhibits TNF-alpha-induced expression of adhesion molecules, markers of vascular inflammation and NF-kB in HUVECs, 2) to determine whether the effects of genistein on TNF- alpha-induced monocyte adhesion to endothelial cells, expression of chemokine, adhesion molecules as well as NF-KB activation is mediated through the cAMP/PKA pathway, and whether stimulation of cAMP/PKA cascade by genistein is mediated through G proteins, 3) to investigate whether dietary intake of genistein protects against TNF-alpha-induced vascular inflammation in mice. Fulfillment of the above aims will establish the cellular and molecular mechanisms of action of genistein in its protective effect against vascular dysfunction. PUBLIC HEALTH RELEVANCE: Atherosclerotic vascular disease is a major cause of morbidity and mortality in the industrial world. Our preliminary data indicate genistein, a soybean compound, is a promising agent to protect against vascular dysfunction caused by the proinflammatory cytokine tumor necrosis factor (TNF)-alpha. The elucidation of the molecular mechanism(s) of action of genistein will provide clinically related strategies in protecting against atherosclerotic vascular disease.